A nucleic acid nanostructure built through on-electrode ligation for electrochemical detection of proteins, peptides, and small molecules

通过电极上连接构建的核酸纳米结构，用于蛋白质、肽和小分子的电化学检测

• 批准号: 10266079
• 负责人: Christopher J Easley
• 金额: $ 29.93万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266079
• 关键词: Affinity; Amino Acid Substitution; Antibodies; Antibody Affinity; Architecture; Binding; Biological Assay; Biological Markers; Biosensor; Biotin; Cells; Clinical; Complex; Custom; DNA; DNA Modification Process; Data; Detection; Development; Diabetes Mellitus; Diagnosis; Diffusion; Digoxigenin; Disease; Disease Marker; Drops; Drug Monitoring; Electrochemistry; Electrodes; Epitopes; Exhibits; Fab Immunoglobulins; Funding; Future; Glucose; Health Status; Heart Diseases; Human; Hydrocortisone; Immunosuppression; Immunosuppressive Agents; Label; Laboratories; Lead; Ligation; Measurement; Medical; Methodology; Methods; Microfluidics; Modification; Molecular; Monitor; Nanostructures; Nucleic Acids; Organic Chemistry; Oxidation-Reduction; Peptides; Pharmaceutical Preparations; Physicians; Preparation; Proteins; Reporting; Research; Scheme; Signal Transduction; Streptavidin; Stress; Structure; Surface; Tacrolimus; Techniques; Technology; Temperature; Therapeutic; Time; Work; analog; base; clinically relevant; cost; design; exenatide; experience; feasibility research; health application; improved; innovation; instrumentation; interest; method development; novel; point of care; response; sensor; small molecule; success; temporal measurement; theories; user-friendly

Diagnosis and treatment of medical conditions could be revolutionized by technology capable of rapid and specific quantification of an arbitrary analyte in real time, over a wide concentration range. To quantify wide ranging clinically relevant targets—small molecules, nucleic acids, or proteins—most method development has drifted towards being target-focused and has lacked generalizability. Currently, the toolbox for potential point-of- care (POC) analysis is a conglomerate of methods or specially targeted probes, and measurement of many targets remains inaccessible to anything other than a large clinical laboratory. There is a pressing, unmet need to develop a platform amenable to rapid, quantitative readout of multiple classes of clinically relevant targets. Electrochemical (EC) sensors have attracted renewed interest for biomarker and drug quantification due to low cost and adaptability to the POC. Still, current approaches (aptasensors, steric hindrance assays) are lacking in generalizability or have complex, noncovalent structures that are not amenable to simple, drop-and-read workflow. In this proposal, we describe our recent development of an innovative nucleic acid nanostructure that exhibits unprecedented generalizability. Strong preliminary data shows this same nanostructure capable of quantifying proteins and antibodies (streptavidin, anti-digoxigenin, anti-exendin-4), peptides (exendin-4), and small molecules (biotin, digoxigenin, tacrolimus). The immunosuppressant drug, tacrolimus, can already be quantified in its therapeutic range. Our objective in this funding period is not only to further develop this new and promising technique, but also to develop a fully surface-confined version that allows true drop-and-read assay workflow that is ideal for POC or real-time clinical measurements. In Aim 1, we will expand the utility of the DNA nanostructure, and modification schemes will be adapted to the most efficient means of detecting proteins, peptides, and small molecules. Nine targeted analytes are relevant to stress/heart disease, immunosuppression, and diabetes monitoring. In Aim 2, we will use organic chemistry to make structural modifications to small molecules or peptides appended to anchor-DNA to fine-tune antibody binding equilibria and improve competitive assays for drop-and-read quantification. In Aim 3, we will develop a fully surface-confined sensor architecture for drop-and-read workflow and real-time measurements. Antibody-DNA or Fab-fragment-DNA conjugates will be used for tethering anchor molecules to the surface alongside DNA nanostructures. Finally, Aim 4 studies will develop instrumentation for improved sensitivity and user experience with the assay. The rationale for this research is to enable measurement of clinically relevant analytes previously inaccessible to EC, while providing a generalizable framework for many other future analytes. The proposed work is significant as a first-of-its-kind assay platform, which we expect to lead to an expanded list of future analytes, previously inaccessible to EC. This proposal is thus innovative in both its technological approach and in its human health applications. Preliminary evidence strongly supports feasibility, and the research team has a proven track-record of success.

医疗条件的诊断和治疗可以通过能够快速和有效地治疗疾病的技术来彻底改变。 在宽的浓度范围内对任意分析物进行真实的实时的特异性定量。量化宽 大部分方法的开发都涉及到临床相关的靶点--小分子、核酸或蛋白质， 倾向于以目标为重点，缺乏普遍性。目前，潜在点的工具箱 护理（POC）分析是一种方法或专门针对性的探针的集合，并且测量许多 除了大型临床实验室之外，任何地方都无法找到目标。有一个迫切的，未满足的需求， 开发一个平台，可以快速、定量地读出多种临床相关靶点。 电化学（EC）传感器由于其低的生物活性而吸引了对生物标志物和药物定量的新的兴趣。 成本和对POC的适应性。尽管如此，目前的方法（适体传感器，空间位阻测定）缺乏在生物学中的应用。 一般性或具有复杂的，非共价结构，不适合简单的，drop and read 工作流在这个提议中，我们描述了我们最近开发的一种创新的核酸纳米结构， 展现出前所未有的普遍性强有力的初步数据显示，这种纳米结构能够 定量蛋白质和抗体（链霉亲和素、抗地高辛、抗毒蜥外泌肽-4）、肽（毒蜥外泌肽-4），和 小分子（生物素、地高辛、他克莫司）。免疫抑制剂他克莫司已经可以 在其治疗范围内定量。我们在这段资助期内的目标，不仅是进一步发展这一新的， 有前途的技术，但也要开发一个完全表面限制的版本，允许真正的滴读分析 这是POC或实时临床测量的理想工作流程。在目标1中，我们将扩展DNA的效用 纳米结构，和修改方案将适用于最有效的检测蛋白质的手段， 肽和小分子。九种靶向分析物与应激/心脏病、免疫抑制、 糖尿病监测。在目标2中，我们将使用有机化学对小分子进行结构修饰， 附加到锚定DNA上的分子或肽，以微调抗体结合平衡并提高竞争性抗体结合平衡。 用于滴读定量的分析。在目标3中，我们将开发一种完全表面限制的传感器架构 用于拖放读取工作流程和实时测量。抗体-DNA或Fab-片段-DNA缀合物将 可用于将锚分子拴系到DNA纳米结构旁边的表面。最后，目标4研究将 开发仪器，以提高检测的灵敏度和用户体验。这样做的理由 研究的目的是能够测量以前EC无法获得的临床相关分析物， 为许多其他未来分析物提供了一个可推广的框架。这项拟议中的工作作为同类第一项工作具有重要意义 分析平台，我们预计这将导致未来分析物的扩展列表，而EC以前无法访问这些分析物。 因此，这一建议在技术方法和人类健康应用方面都具有创新性。 初步证据有力地支持了可行性，研究团队有着成功的成功记录。